Difficulty in aligning small, low-SNR particles (protein-oligonucleotide complex)

Hi Joonyoung,

Re-reading your initial post:

I would be careful doing this. While there is nothing intrinsically wrong with using model-derived templates, it does mean that the visual appearance of classes may be misleading - you will get classes that look like the molecular envelope of your protein, even if you pick from entirely random noise (cf Einstein from noise effect). If you saw clear high resolution features I wouldn’t be worried (because your templates are filtered to 20 Å), but given that features are ambiguous I would maybe reconsider the picking approach.

I would always recommend for an initial analysis using some kind of unbiased approach - either manual picking or blob-based picking, which will usually give good results if appropriately tuned. Subsequently you can take your best subset of 1000-3000 particles and train a Topaz model, which will often give improved results with small/heterogeneous particles.

Cheers
Oli

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